Dicerna Ends Year on Positive Note

With the Notice of Allowance of a US patent application covering its basic Dicer-substrate RNAi trigger technology and a $5M milestone payment from Kyowa Hakko Kirin (KHK) due to the formal initiation of development work for a Dicer-substrate oncology candidate by its Japanese partner, Dicerna can look optimistically into the New Year. These events also symbolize some of the important shifts that have taken place over the last 3 years, namely the increasing choice between RNAi trigger technologies and the increasing importance of Asia in the industry.

Dicer-substrate patent

The patent that is about to formally issue covers methods related to knocking down genes in mammalian cells using certain 25-30 base-pair double-stranded RNAs that undergo Dicer processing before incorporation into the RISC gene silencing complex and target cleavage. While not gate-keeping for Dicer-substrates, the patent will give Dicerna considerable clout over this particular gene silencing approach and has an expiration of at least 2025 if not a year or two longer following patent term adjustments. It is interesting to speculate that the Allowance was also a trigger for the $5M milestone payment.

New oncology candidate formulated in KHK delivery tech

The press release on the $5M milestone noted that while the RNAi trigger of the candidate that is at the center of the $5M milestone payment is licensed from Dicerna (possibly targeting KLF5), the drug delivery tech would be KHK’s own. Based on comments made in early 2010 when Dicerna and KHK first announced their collaboration plus quite a few liposome-related patent apps by KHK, it is likely that the delivery tech relates to a cationic liposomal formulation. I would be a bit wary though about the novelty and strength of these liposomes since at least the patent apps merely claim cationic LNP formulations of chemical compositions that have been known for years in the art an manufacturing method that may be somewhat unique to KHK, but maybe too complex for industrial applicability. Moreover, the described approach lacked the scientific rigor that I would expect of a company that intends to enter clinical development soon.

Dicerna employs Tekmira SNALP delivery

Dicerna has also been saying to be developing delivery technologies, including LNPs and another (actively) targeted delivery system. Interestingly, in an analytical assay development paper that has just been published (Jiao et al., 2011), Dicerna employed Tekmira’s SNALP delivery technology. It is unclear whether the SNALPs were reproduced based on the literature or whether there has been some sort of collaboration between the two companies. However, since Tekmira has been investigating a variety of non-Alnylam RNAi trigger technologies, the latter hypothesis is not that farfetched. The Allowance should provide further impetus for the two companies to work together more closely.

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Gradalis Swiftly Moves ddRNAi-Enhanced Cancer Vaccine Candidate through Clinic

I’ve been reminded a number of times by the staunch Benitec-supporters here that Texas-based biotech company Gradalis has been moving a ddRNAi-enhanced cancer vaccine candidate (‘FANG’) aggressively through clinical development. Virtually out of nowhere, Gradalis initiated clinical trials two years ago and there are now two active phase II trials, one in ovarian cancer and one for advanced melanoma. A peer-reviewed publication on the phase I trial was also just published (Senzer et al.) arguably making FANG the lead RNAi candidate in oncology.

The phase I study involved over 40 patients with advanced solid tumors and demonstrated the safety and logistic feasibility of the approach. Although evidence of suggestive of efficacy was presented such as a clear correlation between an immune response and survival, it would be premature to conclude anything with regard to efficacy. Having now followed a number of cancer vaccines, most of which have eventually failed, it seems to me that correlations such as this could be just as well as a reflection of the fact that those with more responsive immune systems will do better anyway.

‘FANG’ comprises of plasmid DNA from which a single RNA polymerase II promoter drives the expression of an upstream GM-CSF open-reading-frame followed by a pair of downstream RNAi hairpins. This plasmid is introduced by electroporation in a petri dish into the patients’ own cancer cells which have been obtained from a tumor resection. After allowing some time for the expression of the transgenes, the cells are irradiated so as to kill off their proliferative potential and are then re-introduced like many other vaccines by intradermal injection.

The GM-CSF component, wildly popular in the cancer vaccine field and also part of Dendreon’s famous prostate cancer vaccine PROVENGE, is supposed to serve as an attractant, proliferation and maturation factor for dendritic cells which are supposed to ingest, present and thereby stimulate an immune response against the antigens unique to a tumor; the pair of ‘bifunctional’ hairpins meanwhile both target furin which is thought to be an important protease for the maturation of the various isoforms of TGF-beta, a well-known immunosuppressant often overexpressed in cancer.

‘Bifunctional’ here means that one hairpin is perfectly matched and therefore mostly relies on the so-called Ago2/cleavage-dependent mode of RISC activation, whereas the other hairpin contains a central bulge due to mismatching changes introduced in the passenger strand arm of the hairpin thus relying on the non-cleavage pathway of RISC activation which can be facilitated by all four human Argonautes (both predicted to yield the identical guide strand). This strategy of distributing the RNAi between the various Argonaute proteins is certainly an interesting idea, but I’m not sure whether even Gradalis knows what consequences of this is both in terms of efficacy and safety.

A general lack of detailed molecular mechanistic studies is probably my biggest concern with this candidate and when thinking about Gradalis in general. It also at least partly explains why FANG has been moving so rapidly through the clinic. I find it particularly troubling that I have seen no detailed studies by Gradalis looking at the relationship between furin knockdown and TGFbeta inhibition which is key for Gradalis' strategy. This already has caused difficulties in interpreting some of the phase I data where possible assay problems complicated reconciling apparently only modest reductions in furin with much more pronounced down-regulations of TGFbeta. This not only makes it more difficult to make the right development decisions, but also when it comes to finding a partner for the program. On the other hand, you could argue that a cancer vaccine candidate involving both GM-CSF expression and TGFbeta inhibition already has a good chance at succeeding, and sweating out the technical details would only cause delays without making us much the wiser.

As I had mentioned, the Benitec supporters are following Gradalis’ development with much interest as such an advanced ddRNAi candidate may be a prime licensing opportunity for Benitec which controls an important part of the ddRNAi patent landscape. I’ve certainly looked at the hairpin structures involved in light of Benitec’s patent claims (esp. the ‘099 Graham patents) and there is a good chance that Gradalis ought to take a license as it further monetizes this candidate, although their structures may give them a bit of wiggle room.

Mirna Therapeutics selects Marina Biotech’s SMARTICLE delivery tech

In another notable development last week, cancer microRNA Therapeutics company Mirna Therapeutics said that it would use Marina Bio’s SMARTICLE liposomal delivery technology for the development of microRNA replacement therapy for cancer. Based on conference presentations, the two companies had been collaborating on the delivery of microRNA mimics before announcing the deal. An attraction of the SMARTICLE delivery technology, which Marina had acquired from Novosom, is certainly the fact that there is already clinical experience after SMARTICLE-enabled ‘DNAi’ compound by ProNAi has begun dosing a year ago. Similar to related agreements between Mirna and Silence, and InteRNA and Silence, insightful details about the financials were not disclosed. For Marina, which have diluted shareholders by what seems like a 100-fold over the last 3 years (unreal, really), it is good news as its extensive technology offering is finally getting takers.

Mr. Anonymous Thwarted in Zamore US Patent Re-exam

Silence Therapeutics reported that a number of valuable RNAi trigger patents related to the Zamore Design Rules that were issued last year in the US were upheld following a re-examination request by an anonymous 3^rd party. Even stronger claims related to the same Zamore patent series have been issued in Europe, and unsurprisingly Alnylam, but also Novartis and Alcon are opposing them with the outcome to be decided (EP 1633890 B1). However, given the importance of both the US and Europe in the commercialization of innovative drugs, having a patent position in one jurisdiction alone can be considered a valuable strategic asset already.

The Zamore Design Rule patents are owned by the University of Massachusetts and exclusively licensed to Silence Therapeutics for medical uses. As described in more detail in other blog entries before (here and here), they cover methods of promoting the incorporation of the desired guide strand into the RISC gene silencing complex as well as of enhancing RISC turnover resulting in more effective and selective RNAi knockdown.

Especially the patent covering guide strand selectivity methods (‘thermodynamic end-stability rule’; US 7,750,144) is widely recognized in the art for greatly increasing the likelihood of finding efficacious RNAi triggers and is incorporated in essentially all bioinformatic sequence pre-selection algorithms. It has to be said though that the claims do not cover the entire spectrum of approaches of achieving differential end-stability. They do, however, cover chemical and structural approaches that have been reported by a few companies such as Marina Biotech and Sirna/Merck before. Moreover, because the coverage involves modified nucleotides, there will be the concern that even if such modifications were applied for other purposes (e.g. stability or immune abrogation), they may fall under the patents. Consequently, a company with a promising late-stage candidate may want to take a license instead of taking a chance in an infringement lawsuit.

This makes it the second time within a week (see PKN3 opposition by Alnylam) that important patents by Silence Therapeutics were upheld essentially unchanged following challenges. Alnylam’s management once laughed off the value of the Zamore patents in a conference call following their issuance last year. They obviously considered them serious enough to oppose them in Europe and Alnylam is certainly the most likely identity behind Mr. Anonymous (have your say by participating in the survey on the right). Less likely, but not entirely out of the question would be a Big Pharma company like Novartis which is considering taking a license to the Zamore patents, but first wanted to kick the tires before it did so (patents that have been unsuccessfully challenged are considered stronger).

With all these patent successes, and at least one more likely to come, Silence Therapeutics need to monetize their assets in the form of non-dilutive funding.

Hemophilia Gene Therapy Success Bodes well for ddRNAi Therapeutics

Following years of public scorn and derision, including by Alnylam which in 2006 waved off ddRNAi Therapeutics and gene therapy as ‘dangerous’, gene therapy is back with a vengeance. This week, a consortium of researchers reported in the New England Journal of Medicine that an self-complementary AAV8-delivered Factor IX transgene was able to significantly correct hemophilia B in a small clinical trial.

4 out of the 6 patients treated were able to largely discontinue the standard frequent (often 2-3 times a week) and expensive use of prophylactic recombinant FactorIX protein therapy which accounts for estimated healthcare costs north of $20M a lifetime. This means that even in the limited duration of the trial (6-16 months of follow-up), the low cost of the gene therapy ($30k cost of goods for a treatment that is expected to last many years if not a lifetime) meant that this therapy is already saving money (and improving quality of life). If the results can be confirmed in a larger trial and the side-effects, including transient liver enzyme elevations can be controlled with similar efficiency as in this small one, approval may not be that far away.

The hemophilia results also bode well for ddRNAi Therapeutics. First of all, the study has validated the safety and efficacy of delivering the highly promising AAV vector family by peripheral vein infusion to the liver. This represents progress over a previous hemophilia gene therapy study which employed considerably more invasive direct hepatic infusion. In particular, it is likely that, should the ddRNAi collaboration by Tacere/Pfizer for HepC continue, it would involve not only the same vector family (AAV) and target organ (liver), but also the same serotype (AAV8) and self-complementary genome strategy as employed in this hemophilia trial.

Alnylam General Counsel Has Left Company

According to Linkedin, Alnylam's General Counsel, Philip Chase, has just left the company to become General Counsel of Adimab [note: an earlier version incorrectly stated that P. Chase had been with the company for only a little over a year, while in fact he had been there for 4 years]. This departure comes at a critical time for Alnylam as it is involved in a fierce battle with Tekmira for control over SNALP technology, and in which it has started to lose a number of preliminary battles (defamation suit and the City of Hope PR disaster). It also comes after various, possibly related, recent changes including the VP for Intellectual Property (Donna Ward), the lead attorneys in the VSP02 Interference case (Rothwell Figg), a CFO (Patty Allen), key business development personnel as well as two CSOs.

The strongmen at the helm of the company, the CEO and COO, and BoD, however, have largely remained intact during the upheavals over the past 2 years. In fact, John Maraganore (CEO) and Barry Greene (COO) were just re-'incentivized' in the form of options grants that will pocket them $150k and $100k for each US dollar increase in the company's share price (strike price near 3-year low, after 80% stock decline).

Alnylam program update

Based on a poster presentation at this year’s Amercian Society for Hematology meeting (ASH11), also the remaining target pick under Alnylam’s 5x15™ will be a SNALP-based program that targets a gene in the liver: Tmprss6 knockdown for the treatment of iron overload diseases. As such, all of Alnylam’s core clinical programs are SNALP-based. If you had looked at the pipeline 3 years ago, you would have thought that this was where Tekmira was heading. The challenge now will be to clinically mature the programs to a point (in 2015) where the package will be attractive enough for a company like Genzyme/Sanofi-Aventis to acquire the orphan drug company Alnylam for somewhere between $1.0-1.5B.

This development confirms signals that the company has recently started to send out (Rodman Renshaw investor note and comments in the Piper Jaffray presentation) namely that not only is Alnylam now advertising itself as a product, and not platform company, but that it is likely to be all about gene knockdown in the liver. Long gone seem the days when management predicted Alnylam to be the next Genentech. In light of the confrontation with Tekmira, this path opens up the possibility of a settlement whereby Alnylam is allowed continued access to SNALP for the 5 programs, with Tekmira getting full control over other SNALP applications in and especially outside the liver. As a product company that has committed on its programs, why bother with trying to control a platform which is giving Alnylam nothing but legal headaches.

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What Business Does Alnylam’s Counsel Have at the City of Hope?

The Tekmira and Alnylam dispute is a classical David versus Goliath encounter. By my standards, Protiva/Tekmira clearly deserves credit for developing the industry’s most advanced systemic siRNA delivery technology, SNALP. Alnylam, however, is trying to take all of that away from a company that it knows it stands a good chance at silencing with sheer money and influence.

It therefore struck a nerve when I read ‘Opposition 4’ in the ongoing VPS02-related patent interference filed on November 30 by Protiva/Tekmira (Interference No. 105792). It reveals that Alnylam’s counsel, Rothwell Figg*, went to the City of Hope, the employer of a key technical witness by Tekmira (John Rossi), to gently urge them that John Rossi should perhaps consider to withdraw his testimony (from Opposition 4):

‘For example, Material Fact 6, drafted by Alnylam’s counsel, clearly references “the meeting between representatives of City of Hope and Rothwell Figg.” Indeed, in questioning Dr. Rossi, Alnylam’s counsel appears to have acknowledged rather than disputed the occurrence of one or more meetings between Alnylam’s counsel and Dr. Rossi’s employer.’

And this is apparently what Dr. Rossi was informed of happened in those meeting(s):

‘[Dr. Rossi] Well, I was also informed that there was evidence that was evolved that would be used against my disclosure or my declaration in terms of the credibility of me as a -- basically as a scientist. In other words, I would be contradicting my own words, whether they be – I didn’t know what the source was of where my contradiction was going to be brought up, but it was in something that I had published or in publications or in patent applications or patents. That was all I was told. And I felt that it couldn’t be the case. I felt very strongly.

And in fact, Mr. Huntington asked me if I wasn’t worried about the fact that I could actually invalidate my own patents. I said no, not because I don’t want those to be invalidated, but because I didn’t feel that anything I had said or done would contradict those.’

It reads as if Alnylam’s counsel suggested to the employer of the witness that in following his conscience as a scientist, the employee would hurt the financial interest of his employer (City of Hope is the beneficiary Dr. Rossi’s patents). I’m no lawyer, and have no idea whether such apparent manipulation of the judicial process is legitimate or even legal, but it strikes me as very wrong (and desperate).

This episode though is just the latest in the game of politics Alnylam has been playing with Tekmira. It is high time for it to stop it and instead focus on the science. Otherwise, it risks wasting the opportunities that come with the ongoing clinical validation of Tekmira’s SNALP technology to which it is (still) a licensee.

* As I wrote in a blog entry in October, Rothwell Figg seem to have been replaced as Alnylam's counsel in the Interference, to be replaced by Wilmer, Cutler et al. which are also responsible for the larger SNALP Misappropriation case.

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SNALP RNAi versus RNaseH Antisense for Gene Knockdown in the Liver

Following recent phase I results from ISIS Pharmaceutical’s Factor XI (ASH abstract 12999; addendum: PR on phase I data reported on December 12) and Apo C III programs, there is little doubt left that phoshorothioate-based RNaseH antisense as developed by this company and Santaris can mediate target-specific gene knockdown in the liver in Man. These results confirm the clinical experience with the registrational hypercholesterolemia candidate mipomersen and are corroborated by the impressive HCV results obtained by Santaris’ with its anti-miR122 HCV candidate. Beyond the liver, RNaseH efficacy has been demonstrated for solid cancer (custirsen) and possibly Excaliard’s (now Pfizer’s) anti-scarring candidate. On the other hand, the recent trial termination(s) by Santaris, and the safety profiles of mipomersen and OncogeneX' custirsen highlight some of the challenges facing phosphorothioate antisense technology.

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Therefore, as RNAi Therapeutics have progressed from the basic discovery of its mechanism in mammals 10 years ago to solid proof-of-concept gene knockdown in the form of the ALN-TTR01 results 2 weeks ago, it may be a good time to compare and contrast these two technologies also in light of the fact that, after the RNAi Therapeutics backlash, there is a clear trend towards Big Pharma (and other pharmaceutical companies) opening themselves up again towards antisense, meaning that the 2 technologies are competing for precious non-dilutive funding. For this purpose, I will focus on the liver as the best developed target organ for both these technologies.

20 to 100-fold more antisense required

An obvious advantage of antisense, with about 3x the age of RNAi Therapeutics, is that more is known about its clinical pharmacology. As such, there is good visibility as to how much antisense will be needed to achieve the kind of 50-75% knockdown that will be required for therapeutic outcomes in most cases. Dose has important implications particularly in terms of safety and cost.

Current ‘2^nd gen’ molecules (fully phosphorothioated gapmers) like mipomersen require 200mg oligonucleotide per week. Actually, if 300-400mg would have been better tolerated, the higher dosages would have enhanced the commercial profile of mipomersen considerably. Let’s therefore say 1000mg per month for 2^nd gen RNaseH antisense.

With the higher-affinity ‘2.5 gen’ technologies that are starting to move into the clinic, best exemplified by Santaris’ LNAs which in fact may also symbolize the most potent version, it is expected that clinical dosages can be further reduced. Based on the non-human primate data and clinical dosage regimes, I expect that dosages of around 100mg/week or 500mg per month are feasible in the foreseeable future. Also because of the modifications involved, I would be therefore very surprised if the cost of oligonucleotides for treating a patient over a year would be below $15,000 even at commercial scale.

By contrast, it can be expected that it will take about 0.15mg/kg of siRNA formulated in the ‘2^nd gen’ SNALPs that are now moving into the clinic to achieve the type of once-a-month pharmacology that Tekmira and its licensees are aiming for. If you do the math, that translates into about 10mg per month of siRNA oligonucleotides. Give and take the added costs of the lipids and formulation process, but cheaper nucleotide chemistries involved, this translates into a maybe 50-fold cost of goods difference alone. For some diseases and in some countries, this may be less of an issue, but it will be a factor for others.

Safety

The even larger implications of dosage is the related safety. Although clinical repeat-administration studies with SNALP have yet to be conducted, it seems that with the 2^nd gen SNALP formulations, the main safety challenge with SNALP will be in managing acute hypersensitivity reactions around the time of drug administration. Based on similar issues with intravenously administered biologics such as monoclonal antibodies where e.g. transient immune suppression with steroids is routine (and widely accepted), I believe that infusion-related acute toxicities will be manageable.

What is nice with the pharmacology of SNALP RNAi Therapeutics is that the bulk of the drug that does not hit the target, i.e. gets incorporated into the RISC silencing complex, is rapidly turned over by the body, meaning that drug exposure levels between drug administrations will be extremely low. It is because of this that I am hopeful that the risk of causing liver toxicity, long believed to be the main toxicity challenge for SNALP, is quite limited at dosages of 0.15mg/kg/month.

By contrast, RNaseH antisense do not harness a natural gene silencing mechanism and, in the case of the phosphorothioate-based gen 2 and gen 2.5 antisense, work by saturating the target (and off-target) organs with high levels of the ‘sticky’ phosphorothioated oligonucleotides so that mass action carries enough of them into the cells. Consequently, the exposure of the body to the antisense drug is significantly higher compared to SNALP-delivered siRNAs. Assuming ~100-300mg of antisense oligonucleotide per kg of liver or kidney tissue (e.g. ISIS TTR patent application US 2011/0294868) and single-digit microgram siRNA oligonucleotide per kg of liver tissue (e.g. Landesman et al. 2010), you could argue that the real difference in bioburden between antisense and SNALP RNAi is about ten thousand fold. It also does not take into account that it is the sticky phosphorothioate chemistry that is thought to be responsible for much of the toxicity (interactions and turnover) whereas RNAi triggers employ more natural chemistries. On the other hand, double-strand RNAs are recognized by more innate immune receptors than highly modified, single-stranded oligonucleotides.

Route of administration

Although the subcutaneous administration of SNALPs has been demonstrated (see e.g. Tekmira's ApoB patent) and may become practical with the higher potencies of SNALPs and extracellular matrix-degrading technologies as developed e.g. by Halozyme, antisense is currently more amenable to subcutaneous administration whereas SNALP have to be infused in an institutional setting. This means that, as is the case for essentially all monoclonal antibody drugs, SNALP drugs have to address diseases of considerable unmet medical needs where patients do not perceive a once-a-month trip to the infusion center a huge burden. Maybe Pfizer can't, but I can think of many such diseases. Infusion in an institutional setting also has the advantage that acute toxicities, the main safety challenge for SNALPs, can be well managed through professional supervision, whereas patients that inject themselves with antisense at home may be slightly panicked on seeing redness develop at the injection site or on experiencing ‘flu-like symptoms’ that have been reported to occur at high frequency with antisense (often 1/3 to 1/2 of patients), but has surprisingly been little discussed by ISIS Pharmaceuticals.

Manufacturing

Like route of administration, manufacturing is considered to be a practical advantage of antisense over SNALP RNAi. I agree…in purely practical terms. What is, however, entirely forgotten is that as long as you can deal with the manufacturing complexities, it suddenly gives you an invaluable competitive advantage. How about unlimited market exclusivity? Isn’t one of the lessons that Big Pharma should have learned from the current patent cliff that simple small molecule chemistries are highly vulnerable to generic competition? Isn't this also a major reason for why everybody obsesses about monoclonal antibodies these days, yet is often strangely held against SNALP RNAi? To my knowledge, there are no generics of a nanoparticle-formulated drug.

So in summary, as antisense has reached an inflection point as a slew of clinical data is confirming the early clinical results with mipomersen from 6-7 years ago which demonstrated gene knockdown in the liver, SNALP RNAi is making even faster progress with many of its theorized advantages, especially related to the amount of oligonucleotide required and pharmacology, turning into clinical reality quickly. The race is on. The most likely winners meanwhile are the patients.

SNALP Delivery Keeps On Giving: Tekmira Receives OK for Ebola Clinical Studies

On Monday, Tekmira announced that it has received the Green Light from the FDA to go ahead with clinical studies of its SNALP-enabled biodefense candidate for the treatment of Ebola infection. Tekmira is developing TKM-EBOLA under a $140M contract from the US Department of Defense following spectacular results in non-human primates reported last year in The Lancet. Depending on whether you want to count in the stalled TKM-ApoB program or not, this marks the 5^th or 6^th SNALP-enabled candidate in clinical development, illustrating the strength of this systemic RNAi trigger delivery platform: TKM-ApoB, ALN-VSP02, TKM-PLK1, ALN-TTR01, ALN-PCS02, and TKM-EBOLA.

In other words, 6 of the last 7 systemic RNAi INDs or IND equivalents were for SNALP-enabled product candidates (period: 2008-2011). This plus the unparalleled, strong pre-clinical track record of this platform demonstrating efficient knockdown in the liver, solid tumors, and viral infections not only in rodents, but also a number of non-human primate models supports the notion that Tekmira’s SNALP is the industry’s most advanced and valuable RNAi delivery platform. There are thankfully other promising RNAi delivery technologies lining up behind SNALP, but this is not Lake Wobegon where everybody can be above average

Next Steps for TKM-EBOLA

Since TKM-EBOLA, as a treatment for a disease in which controlled human studies are ethically or practically impossible, is being developed under the Animal Rule, this phase I study will not only have to demonstrate adequate safety, but more importantly yield pharmacokinetic and potentially biomarker data that replicates what is seen in the successful treatment of the pre-clinical animal models of the infection. At the same time, it may be worth trying to test the limits of how long treatment can be delayed after symptom onset in the animal models as a common criticism of these studies is that in the real world it may take some time before Ebola victims are identified and treated. In The Lancet studies, rhesus monkeys received first treatments already 30 minutes after exposure to the virus which may model a needle stick scenario in an Ebola research laboratory, but not exposure of the civilian population e.g. in a subway system. Similarly, achieving similar pre-clinical efficacies with 1mg/kg as with the tested 2mg/kg dose in The Lancet studies may bring it more in line with the clinical SNALP safety experience so far. On the manufacturing front, it would be helpful if Tekmira succeeded in providing SNALP in lyophilized form which would increase its utility in the field.

On the other hand, the fact that the rhesus model seems to closely replicate, if not represent a particularly severe form of the human disease, and the absence of a (experimental) therapy for Ebola that has shown comparable promise, should position TKM-EBOLA well for stockpiling despite any lingering real-world concerns. From an Army point-of-view, as long as it has been shown to be safe and well tolerated in humans, having the most promising treatment as a stand-by for a virus as deadly as Ebola is better than nothing at all, a consideration that may result in stockpiling even before, or in the absence of FDA licensure.

In that regard, TKM-EBOLA will be mainly competing with AVI Biopharma’s morpholino antisense candidate AVI-6002 which is being developed under an essentially identical contract with the DoD. A Nature Medicine paper published last year reported that this candidate was successful in rescuing ~60% of infected rhesus macaques, although this represents a roughly 3-fold increase in risk of dying compared to the highly comparable SNALP studies in The Lancet. Nevertheless, AVI Biopharma still enjoys a slight time advantage as it has already begun phase I safety studies earlier this year. A late-October 2011 update by AVI stated that treatment in the first 5 of 6 dose-escalating cohorts was well tolerated and that the Data Safety Monitoring Board recommended further escalation to the last 9mg/kg cohort. Nevertheless, once years behind the AVI program, Tekmira has done well catching up with the competition.

Importance beyond TKM-EBOLA

Besides representing an invaluable strategic asset for Tekmira (it is earning the company significant hard cash now and revenues from stockpiling may come well ahead of the customary 5-10 years it usually takes a normal drug to navigate the FDA approval maze), the approval of the IND further demonstrates that SNALP is indeed the productive delivery platform that also I have long had hopes for it to be, with applications not only for knockdown in the liver and solid cancers, but also phagocytic cells (an important target cell population for the Ebola indication). It is also a stamp of approval by various regulatory agencies around the world that SNALP (including reliable manufacturing) is fit for clinical development. An IND for ALN-TTR02 and phase I results for ALN-PCS02 are next.

Comment on Roche Partnership with PTC

Roche disclosed today that it has signed a collaboration with PTC Therapeutics for the treatment of Spinal Muscular Atrophy, including a $30M upfront fee for pre-clinical assets. This follows a similar deal by AstraZeneca and PTC in oncology earlier this year. PTC develops a platform for the modulation of post-transcriptional processes using orally available small molecules.

What is disappointing to me is that these are examples of Big Pharma companies with an interest in RNA Therapeutics (note that AstraZeneca has a relationship with Silence Therapeutics for which a go/no-go is imminent), but which feel more comfortable risking their money on a technology based on phenotypic tissue culture screens with considerable uncertainty as to clinical relevance and the safety risks inherent in modulating very general gene regulatory mechanisms, instead of using the much more straight-forward oligonucleotide approaches. The reason? Oral bioavailability and coziness with small molecule chemistry. The fate of these collaborations will be an important test case of whether putting patient convenience and other marketing considerations ahead of what is the scientifically best approach will bring Big Pharma the desired outcome. Of note, only a few months ago, Genzyme handed back PTC a candidate for the treatment of Duchenne Muscular Dystrophy and Cystic Fibrosis after spending more than $100M on it.

My view: Technical success trumps patient convenience when it comes to diseases as severe as SMA, DMD, or cancer.

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SNALP Works!

The long wait is finally over: Systemic RNAi delivery has been proven in Man. No 'ifs' or 'buts'. Alnylam just announced that Tekmira’s SNALP-enabled ALN-TTR01 reduced target transthyretin (TTR) protein levels in ATTR patients in a dose-dependent manner- without causing an elevation of liver enzymes or serious adverse event (SAE).

Following preliminary evidence in Tekmira’s TKM-ApoB study, this is the long-awaited moment where there is no doubt that successful systemic RNAi delivery has been achieved. With already about half a dozen SNALP-based programs in or very close to the clinic, this result de-risks a major segment of the RNAi Therapeutics pipeline and should stimulate further investments in SNALP-enabled RNAi Therapeutics development as well as provide a boost of confidence to the entire sector.

Today's presentation at the FAP meeting in Kumamoto can be found here. For a more detailed background on ALN-TTR01 for ATTR, please read yesterday’s blog entry.

In the phase I study (single-dose, dose-escalating), 5 patients received 1mg/kg, the highest planned dose. At this dose, there was a mean reduction of 41% in serum TTR levels from baseline. Despite the small patient numbers and natural TTR variability, this was stat significant at p=0.02 relative to placebo. While this level of knockdown may not seem dramatic and may or may not be useful for eventually achieving a therapeutic effect in ATTR patients, it is important to keep in mind that the other liver-targeted SNALP programs employ formulations that should be at least 10x more potent than the one used in TTR01 and probably also slightly better tolerated. As such, this was a stringent test for the safety and tolerability of SNALP technology and bodes well for the commercial potential of particularly the liver-targeted SNALP pipeline following TTR01, including TTR02 for which an IND is expected by year end.

The time course of TTR protein suppression in one patient provided a picture-book example of bona fide RNAi knockdown in Man. Consistent with the now extensive experience with SNALP in pre-clinical animal models, including non-human primates, this patient exhibited a rapid onset of knockdown (63% reduction at 48 hours) which became a peak knockdown of 81% a week after drug administration with 50% suppression still being observable 4 weeks after this single dose. There is no doubt that this was an RNAi-mediated response.

Preclinical repeat dosing studies have shown that in order to maintain the same level of gene suppression, one can reduce the amount of drug given at subsequent doses. This also means that perhaps giving patients two or three loading doses of 1mg/kg within a week or so may allow one to achieve the 70-80% knockdown with ALN-TTR01 not just in select patients.

Importantly, the safety profile seems to exceed even my own expectations. Unless Alnylam will reveal major immune stimulations in the upcoming conference call (at 8.30am Eastern Time; note added in proof 11/23: none were revealed), the only mild-to-moderate adverse reactions seemed to be infusion reactions that were experienced by 3 out of the 23 TTR01-treated subjects and which was well controlled by simply slowing the infusion rate. As the same was seen in the SNALP-enabled ALN-VSP02 study, it seems that this risk factor is indeed well manageable. What is more, even at the high dose of 1mg/kg, there were no signs of liver toxicity as evidenced by increases in liver function tests. As SNALP-RNAi for ATTR will be a chronic treatment, this should be the major safety focus in the future.

Whatever Alnylam decides to do with ALN-TTR01 (I expect it to remain on the shelf as a viable alternative pending TTR02 results), today represents a milestone in the history of RNAi Therapeutics and I do believe that we have seen the bottom in RNAi Therapeutics. Credit belongs to Alnylam for pushing ahead with the SNALP clinical studies. Alnylam has a truly gifted, and in many ways inspirational drug development team, but it is probably also this talent that has made them blind to what they are actually entitled to. My sympathies are therefore with Tekmira today as it has developed (and owns) the delivery technology that is turning out to be quite literally the savior of RNAi Therapeutics: SNALP. Clinical results eventually follow strong science.

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Phase I Study of ALN-TTR01 in Transthyretin Amyloidosis- A Preview

(For a discussion of the phase I results, see here).

Alnylam is about to reveal results from its phase I study with ALN-TTR01 for the treatment of transthyretin amyloidosis (ATTR) at the Nov20-22 FAP meeting in Kunamoto, Japan. This blog provides a brief overview of the rational for RNAi Therapeutics in this disease and the importance of this particular study for the field of RNAi Therapeutics.

ATTR is an autosomal dominant amyloidotic disease due to point mutations (>100 possible) in the TTR gene. These mutations cause protein misfolding and aggregation into fibrils that, depending on which tissues they accumulate in, can cause various organ dysfunctions, most notably polyneuropathy (FAP), cardiomyopathy (FAC), and gastrointestinal/nutritional defects. For those that develop the disease, death is common 5-15 years following the emergence of disease symptoms (usually between 30 and 50 years of age). Although there is genotype-disease phenotype overlap, the most common mutation, Val30Met, strongly predisposes to FAP, while the Val122 is associated with FAC. About 10,000 patients suffer from FAP and 40,000 from FAC. As a rare genetic disease, it occurs in clusters, with FAP cases for example concentrated in Portugal, Sweden, and Japan.

Until recently, the only accepted treatment has been liver transplantation for FAP where removing the source of the mutant TTR in blood serum can reverse the polyneuropathy. Pfizer just got European approval for FAP with its small molecule TTR conformational stabilizer Vyndaqel based on slowing the rate of peripheral neuropathic impairment.

There exists, however, great need for additional therapies as Vyndaqel actually missed the primary endpoint in its pivotal study (the FDA did not accept the NDA for review earlier this year), and because liver transplantation is ineffective for FAC. The latter seems to be due to wildtype TTR still being able to deposit into pre-existing plaques, for example in heart tissue, at a rate that is higher than the turnover of the amyloidotic plaque. In fact, the amyloidotic potential of wildtype TTR is illustrated by the fact that it frequently causes spontaneous amyloidosis in elderly people (senile ATTR).

RNAi Therapeutics Approach to ATTR

The contribution of both wildtype and mutant to disease pathology, the dynamic turnover of plaques, and the fact that TTR knockout mice have the same life expectancy and fertility as their wildtype littermates and are otherwise essentially asymptomatic, makes RNAi Therapeutics a highly attractive treatment approach for this disease. TTR is involved in the transport of vitamin A and thyroxine in the blood, but it appears that in the absence of TTR these carrier functions are compensated for by other carrier proteins in the serum. What is more, essentially all the life-limiting pathologies are caused by TTR that is expressed in the liver, and with Tekmira’s SNALP delivery technology, RNAi can address the relevant gene expression.

TTR01 vs TTR02

One source of confusion that I expect to affect the financial markets tomorrow stems from Alnylam developing two candidates for ATTR, ALN-TTR01 and ALN-TTR02, the difference between the two candidates being in the SNALP lipid composition. TTR01 is the subject of the present trial and is based on an early DLinDMA lipid-containing formulation. It was shown to be effective in knocking down TTR in non-human primates with an ED50 of around 0.3-0.4mg/kg. As the highest dose in the phase I study was 1mg/kg it is reasonable to expect there to be evidence for TTR knockdown in the ALN-TTR01 trial. I should warn, however, that because of the small patient number in each dose cohort and the natural intra- and inter-patient variability of TTR levels in the serum, the pharmacodynamic outcome measure in this trial, the pooled numbers may not give us a straightforward 'stat-significant' answer.

Because of the rapid developments in improving the efficacy and tolerability of SNALP technology, it is therefore almost assured that Alnylam will drop TTR01 and prioritize TTR02 which takes advantage of these developments and for which the filing of an IND is imminent.

Consequently, the importance of tomorrow’s results for the RNAi Therapeutics field lies in providing proof-of-concept for RNAi knockdown following systemic delivery at OKish tolerability. That’s it. It also sets up the results from the phase I studies with ALN-PCS02, the PCSK9-targeting hypercholesterolemia candidate, which are expected to be reported by the end of this year. As PCS02 uses one of the more recent SNALP formulations, this will be the SNALP candidate that has to shine both in terms of knockdown efficacy and safety/tolerability.

ATTR Economics

If you had any doubts as to the commercial potential of ATTR, it is worth noting that Vyndaqel, Pfizer's just-approved TTR drug, is expected to be priced at more than 100,000 Euros per patient year and that most (known) FAP patients are in healthcare systems that will still bear such costs. Pfizer last year paid $200M in upfront considerations for FoldRx, the original developer of Vyndaqel, with another $200M in contingent milestones. Although FoldRx has a mission of developing other protein folding-based drugs, this price tag was essentially for a drug with results from a pivotal trial that failed to meet the primary endpoint and for which approval was far from certain. Considering Alnylam’s cash position, this one registrational drug candidate valued FoldRx higher than all of Alnylam- although you might justify that with the mounting existential risks stemming from Alnylam's alleged theft and misuse of Tekmira's SNALP technology.

Given these economics, it is not surprising that other companies have similarly recognized the commercial potential of ATTR. Importantly, ISIS Pharmaceuticals will also present progress with its antisense candidate for ATTR (ISIS-TTRRx) at the meeting. This candidate entered clinical development in May and is financed by GSK which retains an option for its exclusive license.

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Genable Finds EUR5M for ddRNAi-Gene Replacement Approach

5 million Euros these days is serious money in RNAi Therapeutics. Last week, Irish biotechnology company Genable Technologies announced that it succeeded in raising such funds for its pre-clinical DNA-directed RNAi Therapeutics-Gene Replacement combo for the treatment of rhodopsin-linked autosomal dominant retinitis pigmentosa (RHO-adRP), an inherited retinal degenerative condition leading to blindness.

To see it flow into DNA-directed RNAi Therapeutics at that makes it particularly noteworthy. It illustrates that ddRNAi is somewhat uncoupled from the current depression in the synthetic RNAi Therapeutics arena due to some technical differences which make it in many ways more similar to traditional gene therapy. Gene therapy, of course, is experiencing a revival, fueled by more and more clinical validation. Incidentally, it which follows a similar boom-bust cycle as RNAi Therapeutics is going through now.

One of the great benefits of ddRNAi Therapeutics is that it can be combined with other DNA-directed modalities, especially the co-expression of protein-encoding genes from the same DNA template. As such, it is particularly amenable to treat autosomal dominant-negative diseases in which the affected gene is important for cellular function thereby precluding approaches that eliminate both the mutated and the healthy copies of a gene. Accordingly, one could use a single DNA molecule from which to direct the suppression of endogenous versions of the affected gene (wildtype and mutant forms) and then replace gene function by providing a version of the gene that is immune to the RNAi.

In Genable’s case, AAV vector capacity limitations meant that a mix of two AAV vectors had to be co-administered by subretinal injection, one carrying the ‘immunized’ rhodopsin gene, the other the shRNA against the endogeneous rhodopsin copies. This apparently did not affect much treatment efficacy. Indeed, it was argued that the co-administration approach would actually increase dosing flexibility by being able to adjust the amount of each component independently. On the other hand, it is possible that the use of vectors with larger capacities, such as the lentiviral system that Oxford Biomedica and Sanofi-Aventis are busy exploring for ocular gene therapy, may have its advantages here. Importantly, it was shown that therapeutic efficacy in the retinitis pigmentosa animal models strictly depended on the presence of both gene replacement and gene knockdown components.

Although the concern with causing harm by knocking down the wild-type allele is often only a theoretical one, as is for example the case with Huntington’s Disease (HD), and depends on the degree of knockdown, other applications for which this approach holds promise are sickle cell anemia, alpha-1 antitrypsin, pachyonychia congenita (PC), amonst many others.

The PC project by TransDerm and collaborators is a good example in the RNAi Therapeutics space for the challenges faced by the alternative approach to treating such autosomal dominant diseases, namely by specifically directing the drug candidate towards the mutated gene/gene product so as to leave the wild-type copy untouched. The problem here is that because the potential mutations can be broadly distributed over a gene, restricting a therapeutic to targeting only one such genetic lesion (or associated gene polymorphisms) can dramatically reduce the number of patients eligible for such a targeted therapeutic. This also explains why the first clinical trial by TransDerm was a placebo-controlled, split-body dose-escalation trial in a single patient (!) as there may not be more than a handful of known patients in the world with the applicable mutation.

The 5 million Euros will be used to bring Genable’s lead candidate, GT038, into clinical development. RHO-adRP seems like a good ddRNAi application as even the correction of only a fraction, e.g. 1/3 to 1/2 of the affected photoreceptors, may be enough to support vision. It is good to finally see a number of ddRNAi Therapeutics candidates based on sound pre-clinical data move into the clinic.

(For another promising late preclinical candidate, see Calimmune's HIV approach)

Arrowhead Research Lifts Veil on Recent Progress in DPC Delivery

Four years after Mirus scientists published their seminal paper in polymer-mediated siRNA delivery, and 3 years after the acquisition of the Dynamic Polyconjugate (DPC) technology by Roche, Arrowhead Research, the new owner of the technology, finally lifted the veil on recent progress in bringing DPC technology to the clinic.

The White Paper reveals, for the first time, that DPCs have been successfully delivered to the livers of non-human primates, and moreover that they may also have use for applications outside the liver, one of the theorized attractions of DPCs based on their small size. Reading between the lines, however, it is also possible to identify some of the factors that have delayed clinical translation, and despite the progress and substantial investments by Roche, it remains an open question when the first DPC-based RNAi Therapeutic will enter clinical development.

Short circulation times were one of the deficiencies of 1^st generation DPCs. In order to reach tissues besides the liver, it is critical to achieve circulation times that are long enough so that the drug gets a chance to find and accumulate in its target tissues. Although DPCs on paper seemed to incorporate the features necessary for achieving such long circulation times, it came somewhat as a surprise when a DPC imaging paper last year showed DPC circulation times to be quite poor (Mudd et al., 2010). Although the particles were still able to accumulate in the liver, the data also raised questions whether the causes of the unexpected pharmacokinetics could have other consequences besides impacting biodistribution, for example in terms of safety.

It turns out that the short circulation times were the result of the premature exposure of the chemical groups that were supposed to shield the membranolytic functionalities of the DPCs outside their target cells. This would also explain why there seem to have been toxicity issues not just due to the poor biodegradability of the polymers, but also because such premature exposure renders DPCs as troublesome as many of the positively charged first-generation polymer approaches.

The White Paper indicates that the instability (and biodegradability) issue has been remedied to some degree such that the longer-circulating DPCs now show first promise for delivery outside the liver. But DPCs may also be competitive for delivery to the liver considering the following performance in non-human primates (excerpt from the White Paper):

‘Latest generation DPCs are remarkably efficacious in rats and non-human primates with ED80 values of ~0.1 mg/kg siRNA after a single dose. Increasing the dose two-fold in non-human primates results in >99% knockdown with a duration of effect of nearly 7 weeks. This is a 10-fold increase in efficacy compared to first generation DPCs containing PBAVE polymer. Latest generation DPCs are also better tolerated and have therapeutic indices of >10 in non-human primates as calculated from ED80 and NOAEL values.’

A 99% knockdown with 0.2mg/kg for 7 weeks- I’m impressed! It will be important to publish these data so that it is possible to see which model system was used, whether the 99% knockdown was seen for pretty much the 7 weeks, what the tox/tolerability profile was, the route of administration, and finally an explanation why a simple 2-fold increase in dosage had such a dramatic effect on knockdown efficacy.

The White Paper is also a reminder that for RNAi delivery technologies to be viable, there needs to be efficient scale-up. Apparently, manufacturing was, and possibly still is, a major issue with DPCs. At least the initial formulations had to be purified so extensively such that the yield became unacceptable for clinical translation and commercialization. Besides general liposomal expertise, quality manufacturing, of course, is what has always differentiated RNAi delivery company Tekmira from its competition.

My sense is that DPCs still have the potential to become an important delivery alternative. However, it is also clear that the path of DPCs was a tough one and, in the absence of guidance from Arrowhead Research, I expect additional delays (2-3 years?) before we will see the first DPC-based RNAi Therapeutic candidate in the clinic. This view is possibly shared by Alnylam as $10M in upfront and an increase of about $10M in annual operating costs would have been a small price to pay for Alnylam if DPCs were as advanced as Alnylam’s current systemic delivery workhorse, Tekmira’s SNALPs.

Until then, Arrowhead Resesarch needs to hit the ground running on the business development front given the increase in expenses that come with the 40+ research team in Wisconsin. The $15M facility with Lincoln Park Capital at least provides Arrowhead with increased financial flexibility. Ironically, it is positive clinical data from the RNAi Therapeutics candidates that are based on Tekmira’s SNALPs, DPCs most direct competitor that would greatly aid in that goal by re-igniting interest in the RNAi Therapeutics platform.

RNAi Therapeutics Investors Hoping for a Merry Christmas

I’ve just come back from working at the Starbucks across my street which strongly reminded me that Christmas was just around the corner. Christmas this year in RNAi Therapeutics is synonymous with data releases by Alnylam from its transthyretin amyloidosis (ALN-TTR01; data presentation November 20-22 in Japan) and hypercholesterolemia (ALN-PCS02; release of top-line results by year-end) phase I clinical trials. These have the potential to demonstrate, for the first time, direct and physiologically meaningful target gene knockdown following systemic RNAi delivery, and thereby have the potential to turn around still negative RNAi Therapeutics sentiments and depressed valuations.

Some of the anticipation can already be felt in the form of appreciating share prices of Alnylam and Silence Therapeutics, together with Tekmira the companies most directly exposed to the current RNAi Therapeutics dataflow, and the financial analyst-investment community which have turned noticeably bullish on Alnylam. Only Tekmira, the inventor of SNALP technology that powers ALN-TTR01, ALN-PCS02 and 5 other candidates in or close to clinical development, has not participated in the rally by failing to find investors willing to defend its stock after taking on well-connected Alnylam.

In assessing the data, a primary focus will be on whether dose escalation was able to proceedeup to the highest planned doses (1.0mg/kg for ALN-TTR01 and 0.25mg/kg for ALN-PCS02) and whether, despite the small number of patients at the high dose levels, there are clear signs for target gene knockdown. 50% target gene knockdown in both cases would be reasonable goals, and probably also necessary ones to have the desired impact. In the case of ALN-PCS02 there should also be at least a 30% reduction in ‘bad’ LDL-cholesterol, the intended pharmacologic outcome of a PCSK9-targeting agent. In terms of safety, the absence of grade 3 adverse events or worse would be highly welcome, of course, as we would be the absence of consistent and clinically meaningful innate immune activations.

Santaris’ anti-miR122 HCV Drug Continues to Impress

MicroRNA Therapeutics seems to have found its poster child already with Santaris’ miR122 LNA antagonist for the treatment of HCV. In an oral presentation at The Liver Meeting which is just wrapping up in San Francisco, the company reported robust dose-dependent anti-HCV activity in a phase IIa study, with close to a 3-log mean reduction of HCV RNA from baseline and viral load below detection in 4 of 9 patients at the highest dose of miravirsen (7mg/kg). The corresponding abstract marking a milestone in microRNA Therapeutics by reporting first clinical activity of an microRNA Therapeutic was released in early October (click here for commentary). There is no doubt that this drug candidate works as expected/hoped for, and unless the future of HCV treatment is in all-oral combos, anti-miR122 with its uniquely differentiated mechanism of action looks like a valuable addition to the fast-moving field of HCV care.

Arrowhead Research Acquires Roche’s RNAi Therapeutics Assets

The long wait is finally over. Almost a year* after infamously announcing its retreat from developing RNAi Therapeutics in-house as part of a corporate restructuring, Roche has sold its related assets to Arrowhead Research. That announcement sent shockwaves through the RNAi Therapeutics World as investors and potential licensing partners alike took this to mean that something was very wrong with RNAi Therapeutics technology. This led to a freeze in business development activities and precipitously falling valuations. Today’s news brings some closure to Roche’s sorry RNAi adventures and also promises a number of interesting revelations and developments in the months to come.

Arrowhead Research, of course, is already a well known player in RNAi Therapeutics through its Calando subsidiary. Arrowhead in my mind has suffered, however, from not realizing that in order to be a credible platform company, you actually need real scientists, and not just collect abstract intellectual property. It has always baffled me how Calando attempted to license its RONDEL delivery technology without even having a laboratory to overcome some of the apparent challenges of this technology.

With about 40 scientists that will join the company from Roche’s Madison, Wisconsin, site ('Mirus') this has obviously changed. This, however, also means that one should expect Calando’s RONDEL technology to be de-emphasized as these scientists will be more motivated to develop the Dynamic PolyConjugate (DPC) delivery technology they invented. As a reminder, DPCs have shown promise in mouse models for delivering siRNAs to selected cell types in the liver. It remains to be seen, however, whether this translates into primates, and whether DPCs have use beyond the liver. A recent imaging paper on DPCs suggests that the latter may be more challenging than one had first hoped (Mudd et al., 2010). Manufacturing and scale-up question will likely also need to be resolved first before entering clinical development.

A key question will be whether Alnylam will bless this spin-out or whether it will it try to undermine Arrowhead Research which has now gained access to, amongst other assets, Tekmira’s SNALP delivery technology and Alnylam’s own RNAi triggers. This could make the New Arrowhead a direct competitor of Alnylam, similar to Tekmira. While I am not sure yet whether Arrowhead management shares my view, but given the advancements of SNALP technology in the clinic, whilst DPCs have been stuck in pre-clinical for some time now, it would be obvious for Arrowhead to create near-term value by taking advantage of the well trodden path of advancing SNALP programs into the clinic. Of course, access to some of the newer lipids would be desirable.

For Arrowhead shareholders, this is likely a good deal given the depressed asset valuations in RNAi Therapeutics and Roche’s apparent desire to get out of RNAi Therapeutics. However, Roche will not be out entirely as it obtained a minority stake in Arrowhead and apparently retained licensing options and milestone/royalty streams to future products which are likely to be somewhat punitive considering that Arrowhead Research did not have much cash to offer.

Equally interesting for Arrowhead shareholders is a financing agreement with Lincoln Park Capital (LPC) under which LPC will provide up to $15M in funding over the next 3 years. Sounds familiar? If so, then you have probably seen LPC reach an almost identical deal with Marina Biotech just last week. The benefit for Arrowhead and Marina is that this source of capital brings some much-needed financial stability to the companies, just as Benitec used a similar arrangement with La Jolla Cove Investors to finance its recent transformation. One can expect, however, that LPC does not view their investments as charitable donations, and based on Benitec’s experience, it will both likely serve to support Arrowhead’s share price, but also somewhat cap its upside.

* An earlier version incorrectly referred to the waiting period as two years.

Silence Therapeutics Finds another Cancer MicroRNA Therapeutics Partner

It’s actually very simple, and apparently it is small biotechnology companies that are first to realize and act on it: Develop a technology that can deliver small silencing RNAs to a given cell/tissue type, and only our exploding insights into the genetics of disease set the limit for the number of potential indications. This benefits both the delivery company that can re-coup some of their investments through licensing out its technology for a few of the many possible targets, and also the licensee which does not need to exhaust and risk its capital to develop its own delivery technology, but can focus on their targets and pick something already fairly de-risked off the shelf for what should be reasonable financial terms at this juncture.

This must have been the reason why Mirna Therapeutics, after apparently having given up on neutral lipid emulsion technology it had developed with BIOO Scientific (LANCEr), has now chosen to partner with Silence to evaluate that company’s endothelial cell-directed AtuPLEX delivery system, which has already shown some promising results in the clinic (see ASCO 2011 presentation), and Silence’s more novel DBTC delivery system for hepatic nucleic acid delivery, for use with its MicroRNA Therapeutic payloads (most likely mimics) to treat cancer. This follows similar deals last month with Dutch cancer MicroRNA Therapeutics company InteRNA, and a collaboration with a mysterious ‘Top Ten Pharma’ (most likely Takeda) concerning the AtuPLEX-related DACC delivery system for lung endothelial cell-directed siRNA delivery.

Whether all these deals will pay off for Silence and their partners now depend on their progress in the lab. It is likely that the work with Mirna Therapeutics will involve miR-34 which is a well validated tumor suppressor microRNA, and has also been implicated in angiogenesis, making it an interesting microRNA mimic to be evaluated with Silence’s lipid-based delivery systems.

As I’m writing this, Arrowhead just announced that it has acquired Roche’s RNAi assets…deal activity in RNAi Therapeutics is clearly heating up again!

Liposomes Also Useful for Functional siRNA Delivery to Phagocytic Cells

The goal in developing a RNAi Therapeutics delivery technology is to achieve gene knockdown in a certain cell or tissue type. Once this is achieved, only our exploding genetic insights into disease limit the potential therapeutic applications. This is for example why we are seeing this SNALP-fuelled expansion in the clinical pipeline for indications in which gene knockdown is targeted in the liver.

In addition to the liver (SNALP), vascular endothelial cells (AtuPLEX, DACC), tumor cells (SNALP), skin (self-delivering rxRNAs), hematopoietic stem cells (lentivirus), and cells in the eye and CNS (lentivirus, AAV), RNAi Therapeutics delivery technologies have reached sufficient maturity to warrant their clinical development for knockdown in phagocytic cells of the immune system. In fact, there is already a clinical trial by Duke University that involves the ex vivo electroporation of siRNAs into dendritic cells for therapeutic cancer vaccine development (think Dendreon), and a first systemically administered example, Tekmira’s SNALP-EBOLA, is about to enter the clinic with an IND planned by year-end.

Probably fearing that the investing public has largely forgotten about such potential of Tekmira’s technology, Alnylam this week advertised two liposomal siRNA delivery studies, one published in Molecular Therapy (Basha et al.) and the other one in Nature Biotechnology (Leuschner et al.), which nicely characterized RNAi activity in phagocytic cells and provided a few tanatalizing examples of their potential therapeutic applications.

Leuschner et al: Interfering with Pathologic Phagocyte Recruitment by Knocking Down CCR2

The Leuschner at al. study, stemming from a collaboration between the Massachusetts General Hospital, MIT and Alnylam, used lipidoid lipid-formulated siRNAs to target chemokine receptor CCR2 in phagocytic cells. The lipid was the C12-200 cationic lipidoid that was shown in a paper last year to promote gene knockdown in the liver at much lower dosages compared to 1^st-generation lipidoids (Love at al. 2010).

After demonstrating that the systemically administered liposomes were taken up by various phagocytic cell populations in the bone marrow, spleen, and the circulation, the researchers then focused in, for the functional part of their study, on the Ly-6high subset of these cells from the spleen. These were chosen because of the particularly efficient liposomal uptake in this cell population and their role in the pathogenic, CCR2-dependent recruitment to sites of local inflammation.

As a reminder, while such inflammatory recruitment is a vital part of our first-line defense and response to foreign invaders and tissue damage, respectively, they often end up doing their job too enthusiastically leaving behind pathologic scars, unwittingly promote cancers, or interfere with medical interventions such as transplantation.

Leuschner and colleagues studied four settings where interfering with Ly-6high through CCR2 knockdown might be beneficial (all mouse studies): ischemia-reperfusion injury as would occur following a heart attack; inflammation at atherosclerotic lesions; pancreatic islet graft survival; and reducing the number of tumor-associated macrophages which are thought to promote cancer. In all four settings, it was found that a moderate ~50% knockdown of CCR2 was sufficient to effectively inhibit inflammatory cell recruitment to the sites of disease and was accompanied by the hoped-for outcome.

While this is certainly encouraging news and seems to open up many therapeutic avenues just based on CCR2 targeting, the degree of knockdown suggests that while it should be possible to achieve therapeutic outcomes with well-chosen target genes, further improvements in efficacy are necessary for the particular formulation to be broadly applicable to other target genes that may not be as dosage-sensitive as CCR2 appears to be.

Basha et al.: Tekmira SNALPs Promote Gene Knockdown in Antigen-Presenting Cells

Unlike the Leuschner et al. study which focused on the potential therapeutic applications of gene knockdown in phagocytic cells, the Basha et al. studied the mechanism, efficacy, and safety of SNALP delivery to antigen-presenting cells (APCs). This paper was largely conducted in the laboratory of Alnylam's new friends from Vancouver.

Evaluating four SNALP formulations differing only in the nature of their ‘critical’ lipid- DlinDAP, DLinDMA, DLinK-DMA, or DLinKC2-DMA- it was found that three of them (DMA, K-DMA, KC2-DMA) were capable of promoting gene knockdown in antigen-presenting macrophages and dendritic cells- both in vitro and in vivo following systemic administration. Taking into account safety, the authors favored the formulation with the KC2-DMA lipid, the lipid of the Tekmira line-of-research that was shown last year in the Nature Biotechnology paper to be highly efficient also for gene knockdown in the liver (Semple et al. 2010).

Importantly, the present data showed that increased efficacy did not come at the cost of safety. Safety is all the more important here since, similar to the lipidoid formulations described above, the delivery efficacy is still much less compared to their use in functional SNALP delivery to the liver, meaning that higher dosages would have to be administered. Since hepatotoxicity could be a dose-limiting toxicity for these formulations, the demonstration that by increasing particle size, gene knockdown in the liver could be abolished without compromising gene silencing in APCs, suggests a strategy to increase the safety of APC-targeted SNALP applications.

The Basha et al. Study Employed Tekmira-Owned Lipids and Formulations and was Partly Funded by Tekmira

While the specific results were not really surprising in light of what has already been known about liposomal delivery, what really confused me was the fact that it was Alnylam that did all the advertising of the Basha et al. study with no word whatsoever from Tekmira. After all, the paper acknowledged Tekmira as a funder of the study and based on the Responses by Alnylam, including their Canadian subsidiary Alnylam-Canada (aka AlCana), to Tekmira’s allegations, neither Alnylam nor AlCana contest that all four lipids are owned by Tekmira. In fact, KC2 is the latest in the series and, as we know, the Nature Biotech paper in early 2010 was advertised by both companies (see here and here).

The acknowledgement:

‘The authors would like to acknowledge support from the Canadian Institutes for Health Research (CIHR) under U0P grant FRN 59836, as well as support from Tekmira Pharmaceuticals and Alnylam Pharmaceuticals. P.R.C. has a financial interest in Tekmira and receives grant support from Alnylam.’

Sure, Alnylam has acted a bit irrational and confused when it came to Tekmira and its general understanding of what constitutes ownership. However, advertising a lipid for which there has been little controversy that it belongs to Tekmira and a study that was in part paid for by Tekmira, without so much as an acknowledgement of Tekmira sets a new standard. Perhaps as perplexing is the fact that Tekmira has not responded at all to these developments.

I entertain two explanations for this, explanations that I consider equally likely. The first one is that things have gotten so much out of control that Alnylam does not care any more about the liabilities of such press releases. If Tekmira needed to find an example of where Alnylam falsely advertised Tekmira’s technology as its own, one of Tekmira’s allegations, Alnylam just provided it with the most striking one.

The other explanation is that maybe things are pointing towards a settlement or merger of the two companies. This would explain why Tekmira seems to co-operate with Alnylam by not protesting about last week’s press release. It is also of note that Alnylam, as of October 8, is now represented by a new lead counsel in the ALN-VSP02 patent Interference case. Initially represented by Rothwell, Figg, Ernest & Manbeck, it is now the attorneys from Wilmer, Cutler, Pickering, Hale, and Dorr that have taken charge of the Interference for Alnylam…you guessed correctly, the same law firm representing Alnylam in the Big One with Tekmira. An alignment of these two cases would have always made sense. At this advanced stage of the Interference, however, the change seems somewhat surprising, but would make sense if it is now critical to have the two cases strongly aligned so as not to jeopardize a mutual understanding that may have been reached.